Rapid identification and enumeration of the various components of biological fluids is an important research and diagnostic aim. Minimal processing and handling of samples would contribute to the widespread use of such techniques.
In the case of enumeration of leukocyte subclasses of human blood, the need for improved techniques is especially keen. For example, the usefulness of monitoring CD4+ lymphocyte levels in noting the progression from HIV positive status to AIDS has underscored the need for a fast, inexpensive, and reliable method to analyze patient blood samples.
Landay et al., "Application of flow cytometry to the study of HIV infection," AIDS 4:479-497 (1990) describes the utility of a technique in understanding the biology of HIV infection. Multiple-color flow cytometric analysis can be applied to the study of HIV disease by using various monoclonal antibodies to perform phenotypic analysis of blood samples. This technique is also useful in other immune system determinations, as in evaluating the status of organ transplant or leukemia patients.
Flow cytometry is a well-known technique wherein cells may be characterized and separated based on fluorescent emission. A labeled, mono-dispersed cell suspension travels through a tube in a fine fluid stream and is presented to an excitation beam. The emitted fluorescence of each cell is measured by appropriate detectors and the cells may be split into droplets and sorted according to given parameters by electrical and mechanical means.
Flow cytometry may be used to identify and enumerate specific subclasses of blood cells. For example, in U.S. Pat. No. 4,284,412 Hansen et al., lymphocytes which have been reacted with fluorescently-labeled monoclonal antibodies are separated from red blood cells and presented one by one to a fixed detector in a flow cytometry system. Each cell is characterized by analysis of forward light scatter, right angle scatter, and fluorescence. This method requires complex sample preparation and instrumentation. While flow cytometry has improved assay reliability and reproducibility in this application, it generally cannot directly provide absolute cell counts for lymphocyte subsets. Independent white blood counts and differential white counts are required to calculate absolute cell counts per Unit volume. In the usual flow cytometry practice, in order to distinguish lymphocytes from monocytes and granulocytes, a lymphocyte gate based on forward and side light scatter patterns must be established for each sample.
Flow cytometry is not routinely used for identifying and enumerating lymphocyte subclasses in the presence of red blood cells, although U.S. Pat. No. 4,727,020 Recktenwald provides a contrary example. Removal of the red blood cells, by density-gradient separation or lysing, increases the time, cost and number of blood-handling steps per assay. Additional blood-handling steps increase the potential for exposure to bloodborne infectious agents. As stated above, the resultant data produced by the flow cytometry method is inadequate for some purposes. In order to calculate absolute cell count per unit volume, flow cytometric data must generally be combined with additional data obtained from other methods. Also, because flow cytometers conventionally utilize a fluid stream passing through a small nozzle, they may generate aerosols which pose an additional source of biohazardous materials for laboratory personnel.
An alternative is to fix sample position relative to the excitation beam. For example, in U.S. Pat. No. 4,758,727 and its divisional, U.S. Pat. No. 4,877,966, Tomei et al., a method and apparatus for measurement of low-level laser-induced fluorescence is described. In this invention, a coherent laser beam is passed through a three-dimensional scanner and focused onto a static target. The target is an object such as a monolayer cell culture or tissue section. A beam spot, having a size as small as one micron, is passed back and forth across the target by a scanner whose path and movement rate are computer-controlled. Fluorescent light is gathered by a biased-cut fiberoptic base plate and relayed to a detector positioned on the opposite side of the target from the beam.
U.S. Pat. No. 5,037,207, also granted to Tomei et al., discloses a laser imaging system with enhanced spatial resolution and light gathering efficiency which allows for digital imaging of a target of varying size, dependent upon the data retrieval and storage limitations of the supporting computer system. The system utilizes a novel optical fiber detector assembly and a rapid scan for collection of all light from every laser spot to create a quantitative digital reproduction of the image on the surface of a target.
U.S. Pat. Nos. 5,072,382, Kamentsky, and 5,107,422, Kamentsky et al., disclose an apparatus and method for scanning a cell population with a beam to generate multiparameter optical data based on each cell's specific location. The scan is made of a surface on which cells have been deposited. A background level is estimated for the neighborhood surrounding each cell based on digital data and corrections are made for the background level.
In "Acousto-Optic Laser-Scanning Cytometer," Cytometry 9:101-110 (1988) Burger and Gershman and U.S. Pat. No. 4,665,553 Gershman et al., a laser-scanning cytometer is disclosed. An optical scan is made of a lysed and washed sample in a cuvette by a Bragg cell-controlled scanner. The cuvette is translated in a stepwise fashion in one direction relative to the scanner. The scanner operates in a direction perpendicular to the direction of cuvette translation and the scan occurs along the side of the cuvette. Once a cell is located, a beam optimization algorithm operates to steady the beam on the cell and measurements of forward light scatter, orthogonal light scatter, and fluorescence are made. Then the process is repeated.
In U.S. Pat. No. 5,117,466, Buican et al. describe a fluorescence analysis system in which data from a flow cytometer establish identification criteria used by a confocal laser microscope to virtually sort the cellular components of a sample. Birefringent optics and Fourier-Transform technology are used to visually select and display cells or subcellular structures having the desired spectral properties.
In "Fluorescence Analysis of Picoliter Samples," Analytical Biochemistry 102:90-96 (1980) Mroz and Lechene teach a method of handling picoliter-volume samples to gather fluorescence intensity data. Samples are taken up via syringe in a single siliconized capillary tube with oil between the samples. Measurements are made of an optical fluorescence chamber defined by a pinhole diaphragm, a microscope objective, and the diameter of the capillary tube.
U.S. patents granted to Mathies et al. are also relevant to the field of the present invention. In U.S. Pat. No. 4,979,824, a high sensitivity detection apparatus is described. This apparatus is based on a flow cytometry system and utilizes a spatial filter to define a small probe volume that allows for detection of individual fluorescent particles and molecules. Laser power and exposure time of the sample are chosen for the best signal-to-noise ratio. Real-time detection of photon bursts from fluorescent particles is used to distinguish the number, location or concentration of the particles from background energy.
In U.S. Pat. No. 5,091,652 Mathies et al., a laser-excited fluorescent scanner is revealed for scanning separated samples using a confocal microscope. The sample is preferably separated by and detected from an electrophoresed slab gel, but may also be on a membrane, filter paper, petri dish, or glass substrate. The confocal microscope forms an illumination volume in the gel and the beam is oriented so that background scattering is minimized by the polarization characteristics of the scattered light.
U.S. Pat. No. 5,274,240 also granted to Mathies et al. and a continuation-in-part of the above patent, teaches a laser-excited capillary array scanner. This invention is primarily intended for fluorescence detection from an array of capillary tubes containing samples that have been separated by capillary electrophoresis. The fluorescence detection assembly employs a confocal system to detect fluorescence from the interior volumes of each capillary tube.
The current cytometry art generally requires time-consuming and potentially hazardous sample-handling and component separation steps. It fails to allow for rapid volumetric identification and enumeration of sub-populations of a cell suspension that are present within a mixed population. The techniques of the prior art often require trained personnel.
It is therefore an object of the present invention to provide a quick, simple to use, less expensive, safer, automated apparatus and method for directly obtaining counts of specific cellular subsets in biological fluids in a volumetric manner and which require small volumes of sample and reagent.